Biogenesis of specialized lysosomes in differentiated keratinocytes relies on close apposition with the Golgi apparatus

Intracellular organelles support cellular physiology in diverse conditions. In the skin, epidermal keratinocytes undergo differentiation with gradual changes in cellular physiology, accompanying remodeling of lysosomes and the Golgi apparatus. However, it was not known whether changes in Golgi and lysosome morphology and their redistribution were linked. Here, we show that disassembled Golgi is distributed in close physical apposition to lysosomes in differentiated keratinocytes. This atypical localization requires the Golgi tethering protein GRASP65, which is associated with both the Golgi and lysosome membranes. Depletion of GRASP65 results in the loss of Golgi-lysosome apposition and the malformation of lysosomes, defined by their aberrant morphology, size, and function. Surprisingly, a trans-Golgi enzyme and secretory Golgi cargoes are extensively localized to the lysosome lumen and secreted to the cell surface, contributing to total protein secretion of differentiated keratinocytes but not in proliferative precursors, indicating that lysosomes acquire specialization during differentiation. We further demonstrate that the secretory function of the Golgi apparatus is critical to maintain keratinocyte lysosomes. Our study uncovers a novel form of Golgi-lysosome cross-talk and its role in maintaining specialized secretory lysosomes in differentiated keratinocytes.


Figure S1
. Comparative analyses of the lysosomes of proliferative and differentiated keratinocytes.(A) Time-lapse imaging of differentiated keratinocyte expressing LAMP1-RFP by Airyscan super-resolution microscope at 63X magnification.Arrowheads following the dynamics of small LAMP1 carriers.Two different events from the same cell are presented (top and bottom panel).Scale bar=2.5 μm.(B-C) IFM analysis of proliferative and differentiated keratinocytes co-immunostained for LAMP1 with either LAMP2 (B) or LysoTracker Red (C).Cells were imaged by Airyscan super-resolution microscope at 63X magnification.Insets represent 2.5 times magnified white boxed regions.Arrowheads point to the colocalization between the markers.White stars represent the LAMP1 structures that are negative for LysoTracker Red.Scale bar=10 μm.

Figure S4. GRASP65 mediates Golgi-lysosome contacts in differentiated keratinocytes.
(A) IFM analysis of the control siRNA/GRASP65 siRNA treated proliferative keratinocytes using GRASP65 and LAMP1 co-immunostaining.(B-D) IFM analysis of the respective siRNA treated differentiated keratinocytes (as labelled).Cells were co immunostained for LAMP1 with either GRASP65 (B), or GRASP55 (C), or GM130 (D) and imaged by Airyscan super resolution microscope.Scale bar=10 μm.Insets represent 2.5 times magnified white boxed regions.Scale bar=5 μm.(E) GRASP65 and GRASP55 transcript analysis in respective siRNA treated condition, plotted as fold change.Values of 3 independent experiments were included. (F) Immunoblotting analysis of control siRNA and GRASP65 siRNA treated cells.Blots were probed with GRAPS65 or beta-actin (as internal control).(G) Characterization of the giant LAMP1-positive structures in GRASP65siRNA treated differentiated keratinocytes.Cells were stained for LAMP1 and Cathepsin-D, or Arl8B (1 st and 2 nd panels).In the 3 rd panel, cells were incubated with NBD-glucosylceramide (GC) and lysotracker red.Scale bar=10 μm.The merged insets are presented in Fig. 4E.(H) Volumetric distribution analysis was performed as described in materials and methods.The log10 values were plotted as cumulative fractions.Statistical significance of the volume of the Golgi in control siRNA and GRASP65 siRNA treated cells were calculated by Mann-Whitney non parametric test.

Figure S2 .
Figure S2.Presence of Golgi lysosome apposition in differentiated keratinocytes.(A) IFM analysis of proliferative and differentiated keratinocytes co-stained for LAMP1 with either GM130, p230, TGN46 or expressed ARF1-GFP.Cells were visualized by Airyscan superresolution microscope.The numbers (1-3) on the differentiated keratinocytes represent the magnified regions that are presented in Fig. 2B.Insets represent 2.5 times magnified white boxed regions.Pearson's correlation coefficient (r) and Manders' overlap coefficients (M1 and M2) in proliferative cells were calculated and plotted as mean±s.e.m. **** on the graphs indicates significant difference between M1 and M2 values.n=number of cells used for quantification.Scale bar=10 μm.(B) LAMP1-immunogold labelling of ultrathin cryosections to define lysosomes.Represented lysosomal structures are present in both proliferative and differentiated keratinocytes.Scale bar=200 nm.(C) Comparative Golgi morphology in proliferative and differentiated keratinocytes.Scale bar=200 nm.Black arrows pointing to the Golgi stacks, while arrowheads pointing the Golgi vesicles (GVs).Please note that the LAMP1 immunogold labelled sections were also used to analyze the Golgi morphology.Negative staining of the Golgi apparatus by LAMP1 confirms the specificity of LAMP1 immunolabelling in S2B.(D) Quantification of the number of associated GVs /unit of Golgi in proliferative and differentiated keratinocytes.n=27 Golgi from multiple experiments were quantified.The distribution of the values is shown; error bars: SD. (E) Proliferative and differentiated keratinocytes were labeled for WGA-Alexa flour 594, co-labelled for LAMP1.Cells were visualized by Airyscan super-resolution microscope at 63X magnification.Insets represent 2.5 times magnified white boxed region.Scale bar=10 μm.(F) Loading controls for the SUnSET assay presented in Fig. 2C.The total synthesis and secretion were measured with or without the presence of small molecules.P= proliferative and D = differentiated conditions.UT= untreated, CHX= cycloheximide, GCA= Golgicide-A.Total synthesis and secretion is shown by the lysate and in cell medium/supernatant respectively.(G-H) Proliferative and differentiated keratinocytes were co-immuno stained for LAMP1 with (G) MitoTracker Red or, (H) EEA1.Cells were visualized by Airyscan super-resolution microscope at 63X magnification.Scale bar=10 μm.(I-J) Proliferative and differentiated keratinocytes were coimmuno stained for LAMP1 with either (I) LAMP2 (red=proliferative; blue=differentiated) or (J) DAPI respectively.The localization coefficients (Pearson's correlation coefficient (r) and Manders' overlap coefficients, (M1 and M2) values were plotted as mean±s.e.m. n=the number of cells quantified from at least three independent experiments.

Figure S3 .
Figure S3.GRASP65 surrounds the lysosomes in differentiated keratinocytes.(A) Differentiated keratinocytes expressing GRASP65-GFP and GRASP55-mCherry were imaged by a spinning disk microscope.Arrows and arrowheads represent respectively to the colocalization of GRASP65 with GRASP55 in dissociated Golgi stacks and, unique ring like structures of GRASP65 that are negative for GRASP55.Insets represent 2.5 times magnified white boxed regions.Scale bar=10μm.(B) mRNA expression of GRASP55 and GRASP65 in proliferative and differentiated keratinocytes was measured by semi quantitative-RT PCR.(C) Live cell imaging of proliferative keratinocyte expressing GRASP65-GFP and LAMP1-RFP by Airyscan super-resolution microscope.Scale bar=10 μm.(D) The graph represents % of proliferative cells having at least one GRASP65-GFP ring in live or in fixed state.n=number of cells.(E-F) Quantification of coefficients (Pearson's correlation coefficient, r and Manders' overlap coefficients, M1 and M2) between GRASP65-GFP and LAMP1-RFP in the proliferative keratinocytes in live (E) and, in fixed (F) conditions.The values were plotted as mean±s.e.m. n=number of cells from three independent experiments.(G) IFM analysis of proliferative and differentiated keratinocytes immunostained with LAMP1 and LC3 (G) arrowheads represent to the LC3-positive autophagosomes.(H) Co-immunostaining of endogenous GRASP65 with LAMP1.Cells were imaged by Airyscan super-resolution microscope.Insets represent 2.5 times magnified white boxed regions.Scale bar=10 μm.

Figure S5 .
Figure S5.Differentiated keratinocytes produce specialized secretory lysosomes (A) IFM analysis of differentiated keratinocytes expressing ManII-GFP and LAMP1-RFP.Cells were imaged by Airyscan super-resolution microscope.Insets representing two different regions (white boxes) were magnified 2.5 times and shown separately.Scale bar=10 μm.(B) IFM analysis of the co-expressed ts045VSVG-GFP and LAMP1-RFP.Post 20 hours of transfection, cells were shifted to a 40 0 C incubator with 5% CO2 and incubated overnight.The trafficking of VSVG then tracked at 32 0 C over the time points of 30 min, 60 min and 90 min.At the end of each time point cells were immediately fixed with 4% PFA.Cells were imaged by a Spinning disk microscope at 63X magnification.Scale bar=10 μm.(C) Total protein secretion from the proliferative and differentiated keratinocytes was measured by the SUnSET assay in presence or absence of the lysosomal inhibitor bafilomycin-A1.The total secretion was measured at 0h and 2h.P= proliferative and D = differentiated conditions.UT= untreated, GCA= Golgicide-A., Baf.=bafilomycin-A1.Total synthesis and secretion are shown by the signal in lysate blot and in cell medium/supernatant respectively.(D) IFM analysis of differentiated keratinocytes expressing LAMP1-GFP and galT-RFP in presence of bafilomycin-A1(50nM). Imaging was performed by Airyscan microscope at 63X magnification.Scale bar=10 μm.

Figure S6 .
Figure S6.A functional Golgi maintains keratinocyte lysosomes.(A) Time lapse analysis of proliferative keratinocytes expressing ARF1 WT -GFP and LAMP1-RFP.Live cell imaging was performed by Airyscan super-resolution microscope at 63X magnification.Scale bar=10μm.(B) localization coefficients (Pearson's correlation coefficient, r and Manders' overlap coefficients, M1 and M2) in proliferative cells between ARF1-GFP and LAMP1-RFP were plotted as mean±s.e.m. n= the number of cells quantified from at least three independent experiment.(C) IFM analysis of Golgi morphology of differentiated keratinocytes expressing ARF1 T31N -GFP, immuno stained either for GM130 or GRASP65.Scale bar=10 μm.(D) analysis of lysosome morphology of proliferative keratinocyte co-expressed with ARF1 T31N -GFP and LAMP1-RFP.Scale bar=10 μm.(E) Proliferative keratinocytes were treated with pharmacological inhibitors as described in materials and methods.Cells were fixed and stained for GM130 and LAMP1.Insets represent 2.5 times magnified white boxed regions.Arrows indicate to the Golgi and lysosome morphology in all treatment conditions.Scale bar=10 μm.(F) HeLa cells were transfected with either ARF1-GFP or ARF1 T31N -GFP and immunostained with LAMP1.Scale bar=10 μm.(G) Proliferative and differentiated keratinocytes either treated with DMSO or BFA, fixed and stained for WGA Alexa flour 594 (WGA-A594) and LAMP1.Insets represent 2.5 times magnified white boxed regions.Arrows indicate to the effect of BFA on the plasma membrane binding of WGA.In all images: scale bar=10 μm.